Self-regulated left-right asymmetric expression of Pitx2c in the developing mouse limb

AbstractThe transcription factor Pitx2c is expressed in primordial visceral organs in a left-right (L-R) asymmetric manner and executes situs-specific morphogenesis. Here we show that Pitx2c is also L-R asymmetrically expressed in the developing mouse limb. Human PITX2c exhibits the same transcriptional activity in the mouse limb. The asymmetric expression of Pitx2c in the limb also exhibits dorsal-ventral and anterior-posterior polarities, being confined to the posterior-dorsal region of the left limb. Left-sided Pitx2c expression in the limb is regulated by Nodal signaling through a Nodal-responsive enhancer. Pitx2c is expressed in lateral plate mesoderm (LPM)–derived cells in the left limb that contribute to various limb connective tissues. The number of Pitx2c+ cells in the left limb was found to be negatively regulated by Pitx2c itself. Although obvious defects were not apparent in the limb of mice lacking asymmetric Pitx2c expression, Pitx2c may regulate functional L-R asymmetry of the limb

Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3′ untranslated region (3′-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3′-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3′-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node

The transcription factor FoxH1 (FAST) mediates Nodal signaling during anterior-posterior patterning and node formation in the mouse

FoxH1 (FAST) is a transcription factor that mediates signaling by transforming growth factor–β, Activin, and Nodal. The role of FoxH1 in development has now been investigated by the generation and analysis of FoxH1-deficient (FoxH1(−/−)) mice. The FoxH1(−/−) embryos showed various patterning defects that recapitulate most of the defects induced by the loss of Nodal signaling. A substantial proportion of FoxH1(−/−) embryos failed to orient the anterior-posterior (A-P) axis correctly, as do mice lacking Cripto, a coreceptor for Nodal. In less severely affected FoxH1(−/−) embryos, A-P polarity was established, but the primitive streak failed to elongate, resulting in the lack of a definitive node and its derivatives. Heterozygosity for nodal renders the FoxH1(−/−) phenotype more severe, indicative of a genetic interaction between FoxH1 and nodal. The expression of FoxH1 in the primitive endoderm rescued the A-P patterning defects, but not the midline defects, of FoxH1(−/−) mice. These results indicate that a Nodal-FoxH1 signaling pathway plays a central role in A-P patterning and node formation in the mouse

Role of Ca2+ transients at the node of the mouse embryo in breaking of left-right symmetry

Immotile cilia sense extracellular signals such as fluid flow, but whether Ca2+ plays a role in flow sensing has been unclear. Here, we examined the role of ciliary Ca2+ in the flow sensing that initiates the breaking of left-right (L-R) symmetry in the mouse embryo. Intraciliary and cytoplasmic Ca2+ transients were detected in the crown cells at the node. These Ca2+ transients showed L-R asymmetry, which was lost in the absence of fluid flow or the PKD2 channel. Further characterization allowed classification of the Ca2+ transients into two types: cilium-derived, L-R-asymmetric transients (type 1) and cilium-independent transients without an L-R bias (type 2). Type 1 intraciliary transients occurred preferentially at the left posterior region of the node, where L-R symmetry breaking takes place. Suppression of intraciliary Ca2+ transients delayed L-R symmetry breaking. Our results implicate cilium-derived Ca2+ transients in crown cells in initiation of L-R symmetry breaking in the mouse embryo

Bicc1 ribonucleoprotein complexes specifying organ laterality are licensed by ANKS6-induced structural remodeling of associated ANKS3.

Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3' UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation

Role of Ca2+ transients at the node of the mouse embryo in breaking of left-right symmetry

Immotile cilia sense extracellular signals such as fluid flow, but whether Ca2+ plays a role in flow sensing has been unclear. Here, we examined the role of ciliary Ca2+ in the flow sensing that initiates the breaking of left-right (L-R) symmetry in the mouse embryo. Intraciliary and cytoplasmic Ca2+ transients were detected in the crown cells at the node. These Ca2+ transients showed L-R asymmetry, which was lost in the absence of fluid flow or the PKD2 channel. Further characterization allowed classification of the Ca2+ transients into two types: cilium-derived, L-R-asymmetric transients (type 1) and cilium-independent transients without an L-R bias (type 2). Type 1 intraciliary transients occurred preferentially at the left posterior region of the node, where L-R symmetry breaking takes place. Suppression of intraciliary Ca2+ transients delayed L-R symmetry breaking. Our results implicate cilium-derived Ca2+ transients in crown cells in initiation of L-R symmetry breaking in the mouse embryo

Oligonucleotide primer sequences used in this study.

Oligonucleotide primer sequences used in this study.</p

Left/right patterning defects in Anks3^Δ/Δ mutant mice.

(A) Lungs, hearts, thoracic, and abdominal blood vessels of representative Anks3 Δ/Δ pups and heterozygous control litter mates on postnatal day P0. Schematic depictions of organ patterning are shown below. R, L: right and left pulmonary lobes, respectively. (B) Schematic ventral view of a mouse embryo at E8.0 (left) and WISH analysis of Nodal mRNA expression in Anks3Δ/Δ embryos and wild-type control litter mates at 4- and 7-somite stages (right). Scale bars, 500 μm or 100 μm for the magnified node regions (insets). The Nodal pattern (left, bilateral, or absent) in LPM and the number of embryos corresponding to each phenotype are indicated. (C) dsVenus fluorescence at the node of Anks3Δ/Δ and wild-type control embryos harboring the NDE-Hsp-dsVenus-Dand5 3′ UTR transgene. The dsVenus coding sequence fused to the DNA sequence for the 3′ UTR of mouse Dand5 mRNA is under the control of the mouse Hsp68 promoter and 4 copies of the crown cell-specific enhancer (NDE) of mouse Nodal. Images are representative of at least 6 embryos per genotype at the 3- to 5-somite stage. The graphs represent the global dsVenus intensity and fluorescence ratios on the right versus left side of the node for each genotype. Blue circles indicate individual values. Data are means ± SD. Statistical significance was determined using the Mann–Whitney U test, with ns: nonsignificant, ***p p S1 Raw Values file. LPM, lateral plate mesoderm. WISH, whole mount in situ hybridization.</p

ANKS3 and a specific target RNA compete for Bicc1 binding in vitro.

(A) Western blot analysis of ANKS3-Flag from HEK293T cell extracts before (input) and after pull-down by in vitro reconstituted RNPs of recombinant GST-Bicc1 that were pre-assembled with saturating amounts of the fluorescently labeled Dand5-3′ UTR RNA fragment 66–110 or, as a control for nonspecific binding, fragment 226–270 (*). Coomassie blue staining of GST-Bicc1 protein retained by the beads (bottom panel) and the fluorescence of bound RNA (middle panels) are shown below. The quantification of the amounts of target RNA (*) retained by the GST-Bicc1 beads before and after incubation with ANKS3-Flag is shown below the gels. The values for the amounts of pulled down ANKS3-Flag shown in the histogram to the right were normalized to controls without target RNA (100%). (B) Cartoon summarizing the result shown in panel A. (C) Western blot analysis of ANKS3-Flag before (input) and after pull-down by the SAM polymerization mutant GST-Bicc1 mutD versus WT GST-Bicc1 that were saturated with fluorescently labeled Dand5-3′ UTR RNA fragment 66–110 as in (A). Pull-downs of RNA and ANKS3-Flag were quantified as in (A). Values for the amount of bound RNA are relative to the pull-down by WT GST-Bicc1 (100%). (D) Cartoon summarizing the result shown in panel C. Data are means + SD from 3 independent experiments. ns: nonsignificant, *p p p t test). Underlying data can be found in the S1 Raw Images and S1 Raw Values files. GST, glutathione S-transferase; KH, K-homology; RNP, ribonucleoparticle; SAM, sterile alpha motif; WT, wild-type.</p